The goal of this program is to define the functions of coronavirus replicase gene proteins during virus infection. The mouse hepatitis virus (MHV) replicase is translated from the positive strand RNA genome as an 800 kDa polyprotein, and processed by three viral proteinases to yield 16 mature replicase proteins (nsp1-16), that likely mediate replication complex formation and RNA synthesis, in addition to predicted roles in virus host interaction and pathogenesis. The amino-terminal replicase proteins (nsp1, nsp2, and nsp3) comprise a cassette of group specific proteins that are related by organization, mechanism of proteolytic processing, and group-specific evolution. Neither nsp1 (p28) nor nsp2 (p65) has known functions, while nsp3 (p210) is predicted to be a multifunctional protein that contains two known papain-like proteinases (PLP1 and PLP2) as well as other domains with possible functions in RNA synthesis and replication complex formation. However, apart from in vitro studies of nsp3 proteinase function and determinants, nothing is known of the functions of nsp 3 or its domains during virus infection. The three integrated Specific Aims of this proposal will use reverse-genetic and biochemical studies to define the functions of MHV nsp1, nsp2 and nsp3 in replicase protein processing and viral RNA synthesis during MHV infection in culture. These experiments will result in a comprehensive understanding of nsp1, nsp2 and nsp3 roles in replication, will identify potential targets for interference with virus replication, and provide the foundation for future studies of replicase protein functions in pathogenesis and disease. Finally, similarities between MHV and SARS-CoV in the replicase indicate that the results may have applicability to understanding the genetics and functions of replicase proteins of that important human pathogen.